Wang, Shiyan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chung, Po Yin. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 128-155). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Abbreviations --- p.vii / Thesis Content --- p.xi / List of Figures --- p.xv / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Normal Hematopoiesis --- p.1 / Chapter 1.2. --- Hematological Malignancy and the Aberrant Development of Blood Cells --- p.2 / Chapter 1.3. --- Leukemia and Its Classification --- p.3 / Chapter 1.4. --- Childhood Acute Lymphoblastic Leukemia (ALL) --- p.5 / Chapter 1.4.1. --- Epidem iology --- p.5 / Chapter 1.4.2. --- Causes and Risk Factors --- p.6 / Chapter 1.4.3. --- Molecular Pathophysiology --- p.7 / Chapter 1.4.4. --- Clinical Presentation --- p.9 / Chapter 1.4.5. --- Classification --- p.10 / Chapter 1.4.5.1. --- Immunophenotyping --- p.10 / Chapter 1.4.5.2. --- French-American-British (FAB) Classification --- p.12 / Chapter 1.4.6. --- Diagnosis and Prognosis --- p.14 / Chapter 1.4.6.1. --- Morphological and Cytochemical Analysis --- p.15 / Chapter 1.4.6.2. --- Cytogenetic and Molecular Genetic Characterizations --- p.16 / Chapter 1.4.7. --- Treatment --- p.19 / Chapter 1.5. --- Overview of Epigenetics --- p.21 / Chapter 1.6. --- Concepts ofDNA Methylation --- p.23 / Chapter 1.6.1. --- CpG Islands --- p.23 / Chapter 1.6.2 --- Mechanisms of DNA Methylation --- p.24 / Chapter 1.6.3 --- Physiological Roles of DNA Methylation --- p.28 / Chapter 1.6.4 --- Initiation of Aberrant DNA Methylation --- p.30 / Chapter 1.7. --- DNA Methylation in Tumorigenesis --- p.31 / Chapter 1.7.1. --- Regional Hypermethylation --- p.33 / Chapter 1.7.2 --- Global and Regional Hypomethylation --- p.34 / Chapter 1.7.3 --- Microatellite Instability and Oncogeneic Mutation --- p.35 / Chapter Chapter 2 --- Literature Review --- p.37 / Chapter 2.1. --- Aberrant DNA Methylation in Childhood ALL --- p.37 / Chapter 2.1.1. --- Cell Cycle --- p.39 / Chapter 2.1.2. --- Apoptosis --- p.41 / Chapter 2.1.3. --- Tissue Invasion and Metastasis --- p.42 / Chapter 2.1.4. --- Transcription Factors and Metabolic Enzymes --- p.44 / Chapter 2.1.5. --- Putative Tumor Suppressor Genes --- p.44 / Chapter 2.1.6. --- Chromosome Instability --- p.46 / Chapter 2.2. --- Methodologies in DNA Methylation Analysis --- p.50 / Chapter 2.2.1. --- Principle of Methylation-sensitive Arbitrarily Primed PCR (MS-AP PCR) --- p.50 / Chapter 2.2.2. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.53 / Chapter 2.2.3. --- Cloned Bisulfite Sequencing --- p.55 / Chapter 2.2.4. --- Experimental Use of Demethylating Agents --- p.55 / Chapter Chapter 3 --- Background of Research --- p.58 / Chapter 3.1. --- Current Methylation Studies in Childhood ALL --- p.58 / Chapter 3.2. --- Objectives of Research --- p.60 / Chapter 3.3. --- Study Approach and Experimental Design --- p.61 / Chapter Chapter 4 --- Materials and Methods --- p.63 / Chapter 4.1. --- Clinical Samples and ALL Cell Lines --- p.63 / Chapter 4.1.1. --- Clinical Samples from Pediatric Patients with ALL and Normal Healthy Donors --- p.63 / Chapter 4.1.2. --- ALL Cell Lines --- p.63 / Chapter 4.2. --- Genomic DNA Isolation from Clinical Samples and Cell Lines --- p.64 / Chapter 4.2.1. --- Ficoll Gradient Centrifugation --- p.64 / Chapter 4.2.2. --- DNA Extraction --- p.64 / Chapter 4.3. --- MS-AP PCR --- p.65 / Chapter 4.3.1. --- Methylation-sensitive Restriction Enzyme Digestion of Genomic DNA --- p.65 / Chapter 4.3.2. --- Arbitrarily Primed Polymerase Chain Reaction --- p.66 / Chapter 4.3.3. --- Isolation of Differentially Methylated DNA Fragments --- p.69 / Chapter 4.4. --- Cloning of Differentially Methylated DNA Fragments --- p.70 / Chapter 4.4.1. --- TA Cloning --- p.70 / Chapter 4.4.2. --- Screening of Positive Clones --- p.71 / Chapter 4.4.3. --- Preparation of Plasmid DNA by Alkaline Lysis Method --- p.72 / Chapter 4.5. --- DNA Sequence Analysis of Differentially Methylated DNA Fragments --- p.72 / Chapter 4.5.1. --- Dye-terminator Cycle Sequencing --- p.72 / Chapter 4.5.2. --- CpG islands Analysis of Differentially Methylated Sequences --- p.73 / Chapter 4.6. --- DNA Methylation Analysis --- p.74 / Chapter 4.6.1. --- Sodium Bisulfite Modification --- p.74 / Chapter 4.6.2. --- Combined Bisulfite Restriction Analysis --- p.75 / Chapter 4.6.3. --- Cloned Bisulfite Genomic Sequencing --- p.76 / Chapter 4.7 --- Gene Expression Study --- p.76 / Chapter 4.7.1. --- RNA Extraction from Clinical Samples and ALL Cell Lines --- p.76 / Chapter 4.1.2. --- Reverse Transcription PCR --- p.77 / Chapter 4.7.3. --- Semi-quantitative RT-PCR --- p.78 / Chapter 4.7.4. --- 5-aza-2 '-deoxycytidine Demethylation Treatment --- p.79 / Chapter Chapter 5 --- Results --- p.80 / Chapter 5.1. --- Generation of DNA Methylation Pattern by MS-AP PCR --- p.80 / Chapter 5.1.1. --- Differential Methylation Patterns of Childhood ALL --- p.84 / Chapter 5.1.2. --- Methylation Patterns of B and T lineages Childhood ALL --- p.86 / Chapter 5.2. --- UCSC BLAT Analysis of Differential Methylated DNA Sequences / Chapter 5.3. --- Identification of Candidate Gene --- p.89 / Chapter 5.4. --- Fibrillin 2 --- p.90 / Chapter 5.4.1. --- FBN2 CpG Islands: UCSC BLAT Search Analysis --- p.90 / Chapter 5.4.2. --- Verification ofFBN2 by ALL Cell Lines --- p.91 / Chapter 5.4.2.1. --- Semi-quantitative RT-PCR --- p.91 / Chapter 5.4.2.2. --- COBRA --- p.92 / Chapter 5.4.2.3. --- Cloned Bisulfite Sequencing --- p.94 / Chapter 5.4.2.4. --- Demethylation Treatment Resorted FBN2 mRNA Expression in ALL Cell Lines --- p.98 / Chapter 5.4.3. --- Studies ofFBN2 in Childhood ALL --- p.99 / Chapter 5.4.3.1. --- Methylation Analysis --- p.99 / Chapter 5.4.3.2. --- Semi-quantitative RT-PCR --- p.105 / Chapter Chapter 6 --- Discussion --- p.107 / Chapter 6.1. --- Genome-wide Screening Approach: MS-AP PCR --- p.107 / Chapter 6.2. --- Sample Selection in this Study --- p.109 / Chapter 6.2.1. --- MS-AP PCR --- p.109 / Chapter 6.2.2. --- Methylation Studies --- p.109 / Chapter 6.2.3. --- Studies in ALL Cell Lines --- p.110 / Chapter 6.3. --- Methylation Patterns in Childhood ALL --- p.111 / Chapter 6.4. --- Candidate Genes Selection Strategies in MS-AP PCR --- p.112 / Chapter 6.5. --- Fibrillin 2: mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.1 --- ALL Cell Lines --- p.113 / Chapter 6.5.2 --- Childhood ALL --- p.113 / Chapter 6.5.2.1 --- mRNA Expression and Methylation Studies --- p.113 / Chapter 6.5.2.2 --- Statistical Analysis --- p.115 / Chapter 6.5.3. --- Possible Roles of FBN2 in Leukemogenesis --- p.116 / Chapter 6.6. --- Clinical Application of FBN2 Aberrant Methylation --- p.119 / Chapter 6.6.1. --- Tumor Markers --- p.119 / Chapter 6.6.2. --- Use of Demethylating Drugs in Chemotherapy --- p.121 / Chapter 6.7. --- Limitations of Methylation Studies --- p.122 / Chapter 6.7.1. --- MS-AP PCR --- p.122 / Chapter 6.7.2. --- Techniques Used in Methylation Study --- p.122 / Chapter 6.7.3. --- Problems in Methylation Study --- p.123 / Chapter 6.8. --- Future Studies --- p.125 / Chapter Chapter 7 --- Conclusion --- p.127 / References --- p.128 / Appendix --- p.155

Cheung Kin Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-105). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.vi / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.iv / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- Multiple myeloma --- p.3 / Chapter 2.2 --- Epidemiology of MM --- p.3 / Chapter 2.3 --- Risk factors --- p.4 / Chapter 2.4 --- Pathophysiology of MM --- p.5 / Chapter 2.5 --- Clinical presentations and diagnosis --- p.6 / Chapter 2.5.1 --- Diagnosis --- p.6 / Chapter 2.5.1.1 --- Laboratory testing of blood and urine --- p.6 / Chapter 2.5.1.2 --- Radiographic evaluations --- p.1 / Chapter 2.5.1.3 --- Bone marrow biopsy --- p.7 / Chapter 2.6 --- Staging and classification --- p.9 / Chapter 2.6.1 --- Staging --- p.9 / Chapter 2.6.2 --- Classification --- p.11 / Chapter 2.6.2.1 --- Monoclonal gammopathy of undetermined significance --- p.11 / Chapter 2.6.2.2 --- Asymptomatic MM --- p.12 / Chapter 2.6.2.3 --- Smouldering MM --- p.12 / Chapter 2.6.2.4 --- Indolent MM --- p.12 / Chapter 2.6.2.5 --- Symptomatic MM --- p.12 / Chapter 2.7 --- Treatment --- p.14 / Chapter 2.8 --- Epigenetics: DNA methylation --- p.15 / Chapter 2.9 --- Fundamental aspects of DNA methylation --- p.16 / Chapter 2.9.1 --- CpG islands --- p.16 / Chapter 2.9.2 --- Roles of DNA methylation --- p.16 / Chapter 2.9.3 --- Proposed mechanisms of transcriptional repression mediated by methylation --- p.18 / Chapter 2.10 --- Possible mechanisms to initiate aberrant DNA methylation --- p.21 / Chapter 2.11 --- DNA methylation in tumorigenesis --- p.22 / Chapter 2.11.1 --- Oncogenic point C → T mutation --- p.22 / Chapter 2.11.2 --- Global DNA hypomethylation --- p.23 / Chapter 2.11.3 --- Regional DNA hypermethylation --- p.23 / Chapter 2.12 --- Aberrant DNA methylation in MM --- p.25 / Chapter 2.12.1 --- Self-sufficiency in growth signals --- p.25 / Chapter 2.12.2 --- Evading apoptosis --- p.26 / Chapter 2.12.3 --- Insensitivity to antigrowth signals --- p.26 / Chapter 2.12.4 --- Tissue invasion and metastasis --- p.27 / Chapter 2.12.5 --- Infinite replicative potential --- p.28 / Chapter 2.12.6 --- Genome instability --- p.30 / Chapter 2.13 --- Methodologies of DNA methylation analysis --- p.32 / Chapter 2.13.1 --- Genome wide screening method: MS.AP-PCR --- p.32 / Chapter 2.13.2 --- Combined bisulfite restriction analysis --- p.34 / Chapter 2.13.3 --- Cloned bisulfite genomic sequencing --- p.36 / Chapter 2.13.4 --- Treatment with demethylating agent --- p.36 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.38 / Chapter 3.1 --- MM specimens --- p.38 / Chapter 3.1.1 --- MM samples --- p.38 / Chapter 3.1.2 --- MM cell lines --- p.38 / Chapter 3.2 --- Magnetic cell sorting of CD138-positive plasma cells --- p.39 / Chapter 3.3 --- Isolation of nuclear pellet from PB --- p.41 / Chapter 3.4 --- "DNA extraction from MM cell lines, MM plasma cells and PB" --- p.41 / Chapter 3.5 --- MS.AP-PCR --- p.42 / Chapter 3.5.1 --- Restriction enzyme digestion of genomic DNA --- p.42 / Chapter 3.5.2 --- Arbitrarily primed polymerase chain reaction --- p.42 / Chapter 3.5.3 --- Isolation of differentially methylated DNA fragments --- p.43 / Chapter 3.6 --- Cloning of differentially methylated DNA fragments --- p.46 / Chapter 3.6.1 --- TA cloning --- p.46 / Chapter 3.6.2 --- Heat shock transformation --- p.46 / Chapter 3.6.3 --- Screening of positive clones by PCR --- p.46 / Chapter 3.6.4 --- Alkaline lysis for plasmid DNA preparation --- p.47 / Chapter 3.7 --- MS.AP-PCR sequence analysis --- p.47 / Chapter 3.7.1 --- Nucleotide sequencing --- p.47 / Chapter 3.7.2 --- CpG islands analysis of differentially methylated sequences --- p.48 / Chapter 3.8 --- DNA methylation analysis --- p.48 / Chapter 3.8.1 --- Sodium bisulfite modification --- p.48 / Chapter 3.8.2 --- Combined bisulfite restriction analysis --- p.49 / Chapter 3.8.3 --- Cloned bisulfite genomic sequencing --- p.49 / Chapter 3.9 --- Gene expression analysis --- p.50 / Chapter 3.9.1 --- RNA extraction --- p.50 / Chapter 3.9.2 --- Reverse transcription PCR --- p.50 / Chapter 3.9.3 --- 5'-aza-2'-deoxycytidine treatment --- p.51 / Chapter CHAPTER 4 --- RESULTS --- p.53 / Chapter 4.1 --- Generation of DNA methylation patterns by MS.AP-PCR --- p.53 / Chapter 4.1.1. --- Global methylation content in MM samples and normal PB lymphocytes --- p.56 / Chapter 4.1.2. --- Differential methylation in MM --- p.56 / Chapter 4.2 --- UCSC BLAT analysis of differentially methylated DNA fragments --- p.60 / Chapter 4.3 --- Identification of two candidate genes with downregulated expression --- p.60 / Chapter 4.4 --- Zinc fingers and homeoboxes 2 (ZHX2) --- p.62 / Chapter 4.4.1 --- ZHX2 CpG islands BLAT search analysis --- p.62 / Chapter 4.4.2 --- Hypermethylation of ZHX2 in MM cell lines --- p.63 / Chapter 4.4.3 --- Downregulated expression of ZHX2 in methylated MM cell lines --- p.66 / Chapter 4.4.4 --- Restoration of ZHX2 expression by 5-Aza-dC treatment --- p.67 / Chapter 4.4.5 --- Unmethylation of ZHX2 in primary MM tumors --- p.68 / Chapter 4.5 --- Ring finger protein 180 (RNF180) --- p.69 / Chapter 4.5.1 --- RNF180 CpG islands BLAT search analysis --- p.69 / Chapter 4.5.2 --- Hypermethylation of RNF180 in MM cell lines --- p.70 / Chapter 4.5.3 --- Downregulated expression of RNF180 in methylated MM cell lines --- p.73 / Chapter 4.5.4 --- Restoration of RNF180 expression by 5-Aza-dC treatment --- p.74 / Chapter 4.5.5 --- Methylation of RNF180 in primary MM tumors --- p.75 / Chapter CHAPTER 5 --- DISCUSSION --- p.76 / Chapter 5.1 --- Importance of methylation in MM --- p.76 / Chapter 5.2 --- Genome-wide screening approach by MS.AP-PCR --- p.76 / Chapter 5.3 --- Sample selection in MS.AP-PCR --- p.78 / Chapter 5.4 --- Methylation patterns in MM --- p.79 / Chapter 5.5 --- Candidate genes selection strategies --- p.81 / Chapter 5.6 --- Zinc fingers and homeoboxes 2 --- p.81 / Chapter 5.7 --- Ring finger protein 180 --- p.83 / Chapter 5.8 --- Limitations --- p.84 / Chapter CHAPTER 6 --- CONCLUSION --- p.86 / REFERENCES --- p.87

Conclusion. Taken together, paternal factors carried in ASG secretion affect genomic imprinting of developing embryos. The outcome of research work described here deepens our understanding of the role of ASG in maximizing reproductive performance mediated by regulating the epigenetic marks of the genome and in particular the imprinted genes. / Introduction. Our previous in vivo studies in golden hamster have shown the accessory sex glands (ASG) secretion facilitate the development of embryos to term but the underlying mechanism is still not clear. Since the deleterious effect caused by the lack of sperm exposure to ASG secretion is heritable to developing fetus and even after birth, we hypothesized that the paternal factor carried in ASG secretion may change the epigenetic regulation and in particular the imprinted genes of embryonic genome. / Materials and methods. Golden hamster and ICR mouse were used in this study. Hamster is a well-established animal model to study the effect of individual ASG but the genetic background of hamster is poorly known. To verify the specificity of our molecular probe and antibodies used in hamster, a mouse model was also established. Five groups of male hamsters and two groups of male mice were established by surgical treatment. In hamster, (SH) sham-operated, (VPX) ventral prostate-removed, (TX) all ASG-removed, (VPVX) castrated with ASG-removed except ventral prostate and (VX) castrated with intact ASG were established. In mouse, SH and VPX were established. In single-mating of hamster, male was copulated with female at estrus for 15 min. In double-mating of hamsters, female mated with each male for 10 min each. In single-mating of mouse, male was caged with female for 1 h. Epididymal sperm, uterine sperm, fertilized oocytes, pre-implantation embryos and fetuses at 13 days gestation (E13) were collected. Global DNA methylation of sperm, fertilized oocytes, early embryos and E13 fetuses were investigated by indirect immunofluorescence and DNA dot-blot using antibody against methylated DNA. Using the same technique, histone acetylation at lysine 5 residue was detected in male pronuclei of fertilized oocytes, protamine 1 and 2 content were detected in sperm, DNA methyltransferase 1, 3a and 3b activities were detected in early embryos. The crown-rump length and weight of fetuses were measured. Morphology was also examined under scanning electron microscope. Two sets of co-ordinately regulated but oppositely expressed imprinted genes Igf2/H19 and Dlk1/Gtl2 were investigated. H19 differentially methylated region (DMR) and Gtl2 promoter were examined by bisulfite sequencing in sperm and E13 fetuses. Expression of Igf2 and Dlk1 were examined by in situ hybridization and real-time PCR in pre-implantation embryos and E13 fetuses. / Results. Uterine sperm in VPX and TX groups showed no change of DNA methylation level and protamine 1 and 2 content. Fertilized oocytes in VPX and TX groups showed similar DNA methylation level as SH group in both hamster and mouse. Histone hypoacetylation was observed in male pronuclei of hamster but not in mouse. Early embryos in VPX and TX groups showed abnormal level of DNA methylation and Dnmt3b during embryo development in hamster. Replenishment of ASG secretion to sperm from VPX and TX group by double-mating restored the DNA methylation level to normal in early embryos. E13 fetuses of VPX and TX groups in hamster and VPX group in mouse showed DNA hypomethylation. E13 fetuses of VPX group in hamster showed increase in average crown-rump length and body weight with larger variations between individuals. One E13 fetus of VPX group in mouse showed polydactyly and malformation in the head. Real-time PCR showed abnormal expression of Igf2 and Dlk1 in both pre-implantation embryos and E13 fetuses of VPX and TX groups. Bisulfite sequencing showed hypermethylation of H19 DMR in VPX and TX groups of hamster and hypomethylation of Gtl2 promoter in VPX group of mouse. / "August 2007." / Adviser: Pak Ham Chow. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4739. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 194-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Fung, Man See Joyce. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / Table of Contents / Acknowledgements --- p.I / Abstract --- p.II / 論文摘要 --- p.VII / Table of Contents --- p.X / List of Figures --- p.XIV / List of Tables --- p.XVI / Abbreviations --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.l / Chapter 1.1 --- Biology of HPV --- p.2 / Chapter 1.1.1 --- History --- p.2 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Genome structure --- p.3 / Chapter 1.2 --- HPV and cervical cancer --- p.8 / Chapter 1.2.1 --- Classification of cervical lesions --- p.8 / Chapter 1.2.2 --- Natural history of development of cervical cancer --- p.9 / Chapter 1.2.3 --- Risk factors --- p.11 / Chapter 1.3 --- Prevention of cervical cancer --- p.12 / Chapter 1.3.1 --- Vaccination --- p.12 / Chapter 1.3.2 --- Screening --- p.12 / Chapter 1.3.2.1 --- Pap test --- p.12 / Chapter 1.3.2.2 --- HPV DNA test --- p.13 / Chapter 1.3.2.3 --- Methylation pattern as a novel marker --- p.13 / Chapter 1.4 --- Biology of Methylation --- p.14 / Chapter 1.4.1 --- Definition --- p.14 / Chapter 1.4.2 --- Silencing effect --- p.18 / Chapter 1.4.3 --- Roles in normal development --- p.20 / Chapter 1.5 --- Methylation and human diseases --- p.20 / Chapter 1.5.1 --- Genetic diseases --- p.20 / Chapter 1.5.2 --- Cancers --- p.21 / Chapter 1.5.3 --- Methylation and oncogenic viruses --- p.23 / Chapter 1.5.4 --- Potential of methylation pattern as a novel biomarker of cancer --- p.24 / Chapter 1.5.5 --- Epigenetic therapy --- p.25 / Chapter 1.6 --- Methylation and HPV --- p.25 / Chapter 1.6.1 --- History --- p.25 / Chapter 1.6.2 --- Potential roles in transcription regulation of HPV --- p.26 / Chapter 1.6.3 --- Viral gene methylation --- p.27 / Chapter Chapter 2 - --- "Hypotheses, Objectives and Study Design" --- p.28 / Chapter 2.1 --- Hypotheses --- p.29 / Chapter 2.2 --- Objectives --- p.30 / Chapter 2.3 --- Study Design --- p.30 / Chapter Chapter 3 - --- Materials and Methods --- p.34 / Chapter 3.1 --- Work flow --- p.35 / Chapter 3.2 --- Study subjects --- p.37 / Chapter 3.2.1 --- Invasive cervical cancer group --- p.37 / Chapter 3.2.2 --- Low-grade group --- p.37 / Chapter 3.2.3 --- Cell lines --- p.38 / Chapter 3.3 --- DNA extraction --- p.38 / Chapter 3.4 --- HPV genotyping --- p.39 / Chapter 3.5 --- PCR of HPV16 LCR --- p.39 / Chapter 3.6 --- Sequencing of HPV 16 LCR --- p.42 / Chapter 3.6.1 --- Purification of PCR products --- p.42 / Chapter 3.6.2 --- Cycle sequencing reaction --- p.42 / Chapter 3.6.3 --- Purification of cycle sequencing products --- p.43 / Chapter 3.6.4 --- Sequencer and data analysis --- p.43 / Chapter 3.7 --- Bisulfite modification --- p.43 / Chapter 3.8 --- PCR of bisulfite modified LCR --- p.45 / Chapter 3.9 --- Cloning --- p.48 / Chapter 3.9.1 --- Ligation --- p.48 / Chapter 3.9.2 --- Transformation --- p.48 / Chapter 3.9.3 --- Colony PCR --- p.49 / Chapter 3.10 --- Sequencing of clones --- p.51 / Chapter 3.10.1 --- Purification of PCR products --- p.51 / Chapter 3.10.2 --- Cycle sequencing reaction --- p.51 / Chapter 3.10.3 --- Purification of cycle sequencing products --- p.52 / Chapter 3.10.4 --- Sequencer and data analysis --- p.52 / Chapter 3.11 --- Statistical methods --- p.52 / Chapter Chapter 4 - --- Results --- p.54 / Chapter 4.1 --- Sample selection --- p.55 / Chapter 4.2 --- HPV16 LCR PCR and sequencing --- p.57 / Chapter 4.3 --- Methylation patterns --- p.61 / Chapter 4.3.1 --- Cell lines --- p.61 / Chapter 4.3.2 --- Cancer group --- p.63 / Chapter 4.3.2.1 --- Overview --- p.63 / Chapter 4.3.2.2 --- Methylation pattern of the cancer samples --- p.66 / Chapter 4.3.2.3 --- Methylation pattern of the promoter region --- p.74 / Chapter 4.3.3 --- Low-grade group --- p.76 / Chapter 4.3.3.1 --- Overview --- p.76 / Chapter 4.3.3.2 --- Methylation pattern of the low-grade samples --- p.79 / Chapter 4.3.4 --- Comparison of the methylation patterns of the cancer samples and the low-grade samples --- p.84 / Chapter Chapter 5 - --- Discussion --- p.95 / Chapter 5.1 --- Sequence variations of HPV 16 LCR --- p.96 / Chapter 5.2 --- Methylation patterns of CaSki and SiHa cell lines --- p.98 / Chapter 5.3 --- Methylation pattern of the cancer samples --- p.99 / Chapter 5.4 --- Methylation pattern of the low-grade samples --- p.100 / Chapter 5.5 --- Comparison of methylation patterns of the cancer samples and the low-grade samples --- p.101 / Chapter 5.5.1 --- Promoter region in 3' LCR --- p.102 / Chapter 5.5.1.1 --- SP1 binding site --- p.102 / Chapter 5.5.1.2 --- E2BS3 and E2BS4 --- p.103 / Chapter 5.5.2 --- Silencer region --- p.104 / Chapter 5.5.3 --- Enhancer region in central LCR --- p.105 / Chapter 5.5.4 --- CpG sites within 5' LCR --- p.106 / Chapter 5.6 --- Role of methylation in HPV 16 --- p.107 / Chapter 5.7 --- Potential as novel biomarker --- p.108 / Chapter 5.8 --- Conclusions --- p.109 / References --- p.111 / Appendix A

Kan, Hoi Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 103-124). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / PUBLICATION --- p.xvi / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter Chapter 1: --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.1. --- Epidemiology of HCC --- p.3 / Chapter 1.2. --- Etiology of HCC --- p.3 / Chapter 1.2.1. --- Cirrhosis --- p.4 / Chapter 1.2.2. --- Hepatitis virus --- p.4 / Chapter 1.2.3. --- Plant carcinogens --- p.5 / Chapter 1.2.4. --- Miscellaneous factors --- p.6 / Chapter 1.3. --- Clinical presentation of HCC --- p.6 / Chapter 1.4. --- Existing diagnostic tests for HCC --- p.6 / Chapter 1.4.1. --- Alpha-fetoprotein (AFP) --- p.7 / Chapter 1.4.2. --- Imaging --- p.7 / Chapter 1.5. --- Treatment of HCC --- p.8 / Chapter 1.5.1. --- Surgical Resection and Transplantation --- p.8 / Chapter 1.5.2. --- Tumor Ablation or Embolization --- p.8 / Chapter 1.5.3. --- Chemotherapy and Radiotherapy --- p.9 / Chapter 1.6. --- Tumor marker development for HCC detection --- p.10 / Chapter 1.6.1. --- Oncofetal antigens and glycoprotein antigens --- p.11 / Chapter 1.6.2. --- Enzymes and isoenzymes --- p.12 / Chapter 1.6.3. --- Growth factors --- p.12 / Chapter 1.6.4. --- Genetics and epigenetics - mRNA and methylation --- p.13 / Chapter Chapter 2: --- Hypermethylation of tumor suppressor genes in cancer --- p.14 / Chapter 2.1. --- Cancer epigenetics --- p.14 / Chapter 2.2. --- DNA methylation in normal cells --- p.15 / Chapter 2.3. --- Physiological role of DNA methylation in normal cells --- p.18 / Chapter 2.4. --- Aberrant DNA methylation in cancer --- p.19 / Chapter 2.4.1. --- DNA hypomethylation in cancer --- p.20 / Chapter 2.4.2. --- DNA hypermethylation in cancer --- p.20 / Chapter 2.5. --- Development of methylation markers in tumor diagnosis --- p.21 / Chapter 2.5.1. --- Methods for the analysis of DNA methylation markers --- p.22 / Chapter 2.5.2. --- Detection of tumor-associated methylated DNA in the circulation of cancer patients / Chapter 2.6. --- Aim of thesis --- p.27 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.28 / Chapter Chapter 3: --- Methods for detecting DNA methylation --- p.29 / Chapter 3.1. --- Subject recruitment --- p.29 / Chapter 3.2. --- Sample collection and processing --- p.29 / Chapter 3.2.1. --- Tumor tissue samples --- p.29 / Chapter 3.2.2. --- Peripheral blood samples --- p.29 / Chapter 3.3. --- DNA extraction --- p.30 / Chapter 3.3.1. --- Plasma samples --- p.30 / Chapter 3.3.2. --- Blood cells --- p.33 / Chapter 3.3.3. --- Tumor tissue --- p.33 / Chapter 3.4. --- Quantitative analysis of methylated DNA using methylation-sensitive restriction enzyme-mediated real-time quantitative PCR (MSRE-qPCR) --- p.34 / Chapter 3.4.1. --- Methylation-sensitive restriction enzyme-mediated real-time quantitative PCR --- p.34 / Chapter 3.4.3. --- Real-time PCR primer design --- p.36 / Chapter 3.4.4. --- Duplex real-time PCR --- p.40 / Chapter 3.4.5. --- "Real-time detection of GSTP1, SOCS1, A PC, pl6 and ACTB sequences" --- p.41 / Chapter 3.4.6. --- Statistical analysis of real-time PCR results --- p.41 / Chapter 3.5. --- "Methylation study of GSTP1, SOCS1, APC, pl6 and ACTB in tumor tissues and blood cells using bisulfite sequencing" --- p.46 / Chapter 3.5.1. --- Principle of bisulfite modification --- p.46 / Chapter 3.5.2. --- Bisulfite conversion --- p.47 / Chapter 3.5.3. --- Sequencing primer design --- p.47 / Chapter 3.5.4. --- Conventional PCR after bisulfite treatment --- p.49 / Chapter 3.5.5. --- Cloning and bisulfite genomic sequencing --- p.53 / Chapter 3.5.6. --- Data acquisition and interpretation --- p.54 / Chapter SECTION III: --- DEVELOPMENT OF METHYLATION MARKERS IN HCC DETECTION / Chapter Chapter 4: --- Evaluation of the real-time PCR assay for quantification of methylated tumor suppressor genes --- p.57 / Chapter 4.1. --- Development of real-time PCR assays --- p.57 / Chapter 4.2. --- Methylation analyses by bisulfite sequencing were concordant with the real-time quantification results --- p.61 / Chapter Chapter 5: --- Clinical application of methylated markers in the detection of hepatocellular carcinoma --- p.69 / Chapter 5.1. --- Demographics of HCC patients and HB V carriers --- p.69 / Chapter 5.2. --- Quantitative analysis of hypermethylated tumor suppressor genes in tumor and plasma samples --- p.71 / Chapter 5.3. --- Effect of cirrhosis on the plasma methylated tumor suppressor gene concentrations --- p.77 / Chapter 5.4. --- Changes in the concentration of the tumor suppressor genes one month after surgical resection of the cancer --- p.81 / Chapter 5.5. --- Concurrent use of serum AFP level and plasma methylated markers for HCC diagnosis --- p.84 / Chapter 5.6. --- Prognostic value of plasma methylated TSGs --- p.86 / Chapter SECTION IV: --- DISCUSSION --- p.90 / Chapter Chapter 6: --- Discussion --- p.91 / Chapter 6.1. --- Tumor and plasma detection of hypermethylated tumor suppressor genes --- p.92 / Chapter 6.2. --- No effect of cirrhosis on plasma methylated DNA level --- p.94 / Chapter 6.3. --- Clearance of methylated TSG sequences after tumor resection --- p.95 / Chapter 6.4. --- Concurrent use of serum AFP level and the presence of methylated markers in the plasma in HCC diagnosis --- p.95 / Chapter 6.5. --- Prognostic significance of circulating methylated tumor markers --- p.96 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.98 / Chapter Chapter 7: --- Conclusions and future perspectives --- p.99 / REFERENCES --- p.103

Yuen, Ka Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 160-185). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENTS --- p.VI / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Pseudomalignant nature of the placenta --- p.2 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- "Proliferation, migration and invasion behaviour" --- p.3 / Chapter 1.3 --- Gene expression --- p.4 / Chapter 1.3.1 --- Angiogenic factors --- p.5 / Chapter 1.3.2 --- Growth factors --- p.5 / Chapter 1.3.3 --- Proto-oncogenes --- p.6 / Chapter 1.3.4 --- Tumour suppressor genes --- p.8 / Chapter CHAPTER 2: --- Epigenetics --- p.10 / Chapter 2.1 --- Overview --- p.10 / Chapter 2.2 --- DNA methylation in mammals --- p.11 / Chapter 2.3 --- Regulation of DNA methylation machinery --- p.12 / Chapter 2.4 --- Role of DNA methylation --- p.13 / Chapter 2.5 --- Aberrant DNA methylation --- p.16 / Chapter 2.6 --- DNA methylation in normal cells --- p.17 / Chapter 2.6.1 --- X-chromosome inactivation --- p.17 / Chapter 2.6.2 --- Genomic imprinting --- p.18 / Chapter 2.6.3 --- Cell-type-specific methylation --- p.19 / Chapter 2.6.4 --- Placental-specific methylation --- p.20 / Chapter 2.7 --- Aim of Thesis --- p.21 / Chapter SECTION II: --- MATERIALS AND METHODOLOGY --- p.23 / Chapter CHAPTER 3: --- Materials and methods --- p.24 / Chapter 3.1 --- Preparation of samples --- p.24 / Chapter 3.1.1 --- Collection of placental tissues --- p.24 / Chapter 3.1.2 --- Preparation of blood cells --- p.25 / Chapter 3.1.3 --- Preparation of cell lines --- p.25 / Chapter 3.1.4 --- Treatment of JAR and JEG3 with 5-aza-2'-deoxycytidine (5-aza-CdR) and Trichostatin A (TSA) --- p.26 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from tissue samples --- p.26 / Chapter 3.2.2 --- DNA extraction from blood cells --- p.29 / Chapter 3.2.3 --- RNA extraction from cell lines --- p.30 / Chapter 3.3 --- Methylation analysis --- p.31 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.31 / Chapter 3.3.2 --- Bisulfite Conversion --- p.32 / Chapter 3.3.3 --- Primer design for methylation-specific polymerase chain reaction / Chapter 3.3.4 --- Methylation-specific polymerase chain reaction (MSP) --- p.33 / Chapter 3.3.5 --- Primer design for bisulfite sequencing --- p.34 / Chapter 3.3.6 --- Cloning and bisulfite genomic sequencing --- p.35 / Chapter 3.4 --- Quantitative measurements of nucleic acids --- p.39 / Chapter 3.4.1 --- Principles of real-time quantitative PCR --- p.39 / Chapter 3.4.2 --- Real-time quantitative MSP --- p.42 / Chapter 3.4.3 --- Real-time reverse transcriptase (RT)-PCR --- p.42 / Chapter 3.5 --- MALDI-TOF mass spectrometry (MS) --- p.43 / Chapter 3.5.1 --- Principle of homogeneous MassEXTEND assay and MALDI-TOF MS --- p.43 / Chapter 3.5.2 --- Methylation-sensitive restriction enzyme digestion and homogeneous MassEXTEND assay for APC and H19 --- p.46 / Chapter SECTION III: --- A SEARCH FOR HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE HUMAN PLACENTA --- p.48 / Chapter CHAPTER 4: --- Screening on TSGs and non TSGs --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Materials and methods --- p.50 / Chapter 4.2.1 --- Sample collection --- p.50 / Chapter 4.2.2 --- Sample processing and DNA extraction --- p.50 / Chapter 4.2.3 --- Experimental Design --- p.51 / Chapter 4.3 --- Results --- p.63 / Chapter 4.3.1 --- Identification of hypermethylated TSGs by methylation-specific PCR screening --- p.63 / Chapter 4.3.2 --- Validation of hypermethylated TSGs by bisulfite sequencing --- p.69 / Chapter 4.4 --- Discussion --- p.77 / Chapter CHAPTER 5: --- Methylation status of TSGs in different tissues --- p.80 / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and methods --- p.81 / Chapter 5.2.1 --- Sample collection --- p.81 / Chapter 5.2.2 --- Sample processing and DNA extraction --- p.81 / Chapter 5.2.3 --- Experimental design --- p.81 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- Methylation patterns of TSGs in non-placental fetal tissues --- p.86 / Chapter 5.4 --- Discussion --- p.90 / Chapter SECTION IV: --- FUNCTIONAL IMPLICATION OF HYPERMETHYLATED TUMOUR SUPPRESSOR GENES IN THE PLACENTA --- p.94 / Chapter CHAPTER 6: --- Imprinting checking --- p.95 / Chapter 6.1 --- Introduction --- p.95 / Chapter 6.2 --- Materials and methods --- p.96 / Chapter 6.2.1 --- Sample collection --- p.96 / Chapter 6.2.2 --- Sample processing and DNA extraction --- p.97 / Chapter 6.2.3 --- Experimental design --- p.97 / Chapter 6.3 --- Results --- p.100 / Chapter 6.3.1 --- Imprinting checking of H19 by enzyme digestion on placental tissues --- p.100 / Chapter 6.3.2 --- Imprinting checking of　APC by enzyme digestion on placental tissues --- p.101 / Chapter CHAPTER 7: --- CORRELATION OF HYPERMETHYLATION AND GENE EXPRESSION --- p.107 / Chapter 7.1 --- Introduction --- p.107 / Chapter 7.2 --- Materials and methods --- p.108 / Chapter 7.2.1 --- Sample preparation and processing --- p.108 / Chapter 7.2.2 --- DNA and RNA extraction from cell lines --- p.108 / Chapter 7.2.3 --- Experimental design --- p.108 / Chapter 7.3 --- Results --- p.111 / Chapter 7.3.1 --- Methylation status of APC in choriocarcinoma cell lines --- p.111 / Chapter 7.3.2 --- Demethylation of APC in choriocarcinoma cell lines --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Chapter SECTION V: --- CONSERVATION OF METHYLATION IN PLACENTA ACROSS DIFFERENT SPECIES --- p.118 / Chapter CHAPTER 8: --- Methylation analysis of hypermethylated TSG homologues in the placentas of the mouse and rhesus monkey --- p.119 / Chapter 8.1 --- Introduction --- p.119 / Chapter 8.2 --- Materials and methods --- p.120 / Chapter 8.2.1 --- Sample collection --- p.120 / Chapter 8.2.2 --- Sample processing and DNA extraction --- p.120 / Chapter 8.2.3 --- Experimental design --- p.120 / Chapter 8.3 --- Results --- p.124 / Chapter 8.3.1 --- Methylation status of TSGs in rhesus monkey and murine placental tissues --- p.124 / Chapter 8.4 --- Discussion --- p.136 / Chapter SECTION VI: --- CONCLUDING REMARKS --- p.138 / Chapter CHAPTER 9: --- Conclusion and future perspectives --- p.139 / Chapter 9.1 --- Pseudomalignant nature of placenta at the epigenetic level --- p.139 / Chapter 9.2 --- Functional implication of TSG hypermethylation --- p.140 / Chapter 9.3 --- Significance of hypermethylated TSGs in the placental evolution --- p.142 / Chapter 9.4 --- Clinical implication of TSG hypermethylation --- p.143 / Chapter 9.5 --- Future perspectives --- p.145 / APPENDIX I COMPLETE BISULFITE SEQUENCING DATA FOR HYPERMETHYLATED TSGS --- p.147 / APPENDIX II BISULFITE SEQUENCING DATA FOR PTEN --- p.156 / APPENDIX III BISULFITE SEQUENCING DATA OF LOCI NOT SHOWING HYPERMETHYLATION --- p.158 / REFERENCES --- p.160

There has been an increase in the incorporation of silver nanomaterials into consumer products due to their antimicrobial properties. Therefore there is potential for silver nanoparticles (Ag-NPs) to leach out into the environment during different life-cycle stages of these nanomaterial-containing products. Concern about the toxicity of Ag-NPs has led to investigations into their toxic effects on a variety of organisms mainly using acute and sub-chronic, single-generation exposures. The focus of this project was to understand the effects of long-term continuous multigenerational exposure to AgNO3 and Ag-NPs in both pristine and environmentally transformed forms, on the model organism, Caenorhabditis elegans, a soil nematode. A previous multigenerational C. elegans study, showed increased sensitivity in terms of reproductive toxicity, in response to AgNO3 and Ag-NPs, but not sulfidized Ag-NPs (sAg-NPs), with increasing generations of exposure. The reproductive toxicity persisted in subsequently unexposed generations even after rescue from the exposure. We hypothesized that genomic mutations and/or epigenetic changes were possible mechanisms by which the reproductive toxicity was inherited. We investigated the potential for induction of germline mutations in C. elegans after exposures for ten generations to AgNO3, Ag-NPs, and sAg-NPs using whole genome DNA sequencing. Epigenetic changes at histone methylation markers, (H3K4me2 and H3K9me3), and DNA methylation at adenosine (N6-methyl-2’-deoxyadenosine) were investigated after multigenerational exposure as well as after rescue from the exposure using enzyme-linked immunosorbent assays (ELISA) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), respectively. Expression levels of the genes of methyltransferases and demethylases, associated with the histone methylation markers and DNA methylation, were also examined. Our results for germline mutations reveal no significant differences between the nematodes exposed to AgNO3 or pristine Ag-NPs when compared to controls. The significant increase in the number of transversion was observed only for sAg-NPs. However, a trend toward an increase in the total number of mutations was observed in all Ag treatments with some of those mutations having a predicted moderate or high impact. This potentially contributed towards reproductive as well as growth toxicity shown previously after ten generations of exposure in every treatment.. These results did not entirely support the multigenerational reproductive toxicity observed previously. Epigenetic responses at histone methylation markers revealed opposite patterns between pristine and transformed Ag-NPs with Ag-NPs causing a significant increase while exposure to sAg-NPs resulted in significant decrease in methylation at H3K4me2 mark. The increase in H3K4me2 levels was also inherited by subsequent unexposed generations rescued from Ag-NP exposure. Only sAg-NPs caused a significant decrease in methylation at H3K9me3 mark. Changes in mRNA levels for histone methyltransferases and demethylase corresponded with the histone methylation levels affected by Ag-NPs and sAg-NPs. For DNA methylation, a significant increase was observed only for AgNO3, which was not inherited after the rescue. In conclusion, while germline mutations with a high or moderate impact may affect reproduction, our results do not support this as a mechanism for the heritable increase in C. elegans sensitivity to reproductive toxicity from AgNO3 and pristine Ag-NPs. The epigenetic changes, however, do show partial correlation with the observed reproductive toxicity. The reproductive multigenerational effects of AgNO3 can be attributed to changes in DNA methylation whereas that of Ag-NPs can be attributed to changes in histone methylation. Further studies, focused on the investigation of changes in histone and DNA methylation levels at specific loci using chromatin immunoprecipitation sequencing (ChIP-Seq) and methylated DNA immunoprecipitation sequencing (MeDIP-Seq), respectively, are warranted for a better understanding of the impact of such changes.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3' ends, are preferred targets of DNA methylation linked gene silencing. H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread down-regulation of ZNF gene expression which accounted for 8% of all the down-regulated genes in the arsenical-transformed cells. In summary, these studies associate arsenical exposure with agglomerative DNA methylation of gene family clusters and widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of arsenical-induced carcinogenesis.

It has been known for a long time that the cytosine base can be modified to produce a new nucleotide, identified as 5-methylcytosine (mC). In normal cells, mC is correctly distributed into the genome, but in many diseases including life-threatening cancers, its pattern is profoundly perturbed. In 2009, Anjana Rao, published that certain proteins, known as the TET enzymes, are capable of removing mC by further oxidizing it to 5-hydroxymethylcytosine (hmC). This original article, cited more than 1200 times, has led to a great expansion in our understanding of DNA methylation. Such recent publications expanded this knowledge by showing that the TETs successively oxidize hmC to 5-formylcytosine (fC) and 5-carboxylcytosine (caC). <p>These oxidized methylcytosines have been implicated in several mechanisms of DNA demethylation, including “active” demethylation through base excision repair, and “passive” demethylation via successive rounds of DNA replication. In addition, DNA hydroxymethylation is thought to be involved in a wide range of diseases, and a marked decrease of hmC seems to be a “hallmark” of many cancers. <p>However, little is known about the regulation of their modes of action. It is tempting to speculate that these proteins interact with a plethora of factors to elicit coordinated biological functions. Likewise, they might be regulated by environment, which in certain situations, could alter the hydroxymethylome landscape, and lead to cellular malfunction and diseases.<p>In the first study, we pursued a large, unbiased screen of the TET interactome, and discovered that TET2 and TET3 interact with the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). OGT is a glycosyltransferase that adds N-acetylglucose moieties on various proteins, including histone H2B, expanding therefore the “histone code”. We further discovered that the TET-OGT association seems to enhance OGT activity and to potentiate glycosylation and stabilization of SET1/COMPASS, a complex that is responsible for the global deposition of the H3K4me3 histone mark that “decorates” active promoters. Finally, we could confirm a decreased genome-wide H3K4me3 deposition in a model of acute myeloid leukemia mutated for TET2, suggesting that the TET-OGT link is implicated in Health and Disease.<p>In the second study, we looked at the impact of the environment on TET activity and on cellular hydroxymethylomes. We focused on oxidative stress assaults that are known to be involved in inflammation, a mediator of cancer and neurodegenerative diseases. We observed a significant decrease of hmC in cell lines treated with various oxidant stressors, likely due to a direct inactivation of the TETs catalytic domain. Moreover, gene ontology analysis of differentially hydroxymethylated regions (dhMRs), profiled by deep-sequencing on treated vs non-treated cells, highlighted pathways involved in oxidative stress response. The implication of TETs in oxidative stress response was further emphasized by a decreased proliferation of TET1-depleted cells when they are treated with oxidant stressors. Importantly, those results were confirmed in mice knockout for the major antioxidant enzymes GPx1 and GPx2. <p>In conclusion, the work of this thesis contributed to better understand the modes of action of the TET proteins, through (1) direct interaction with OGT, and (2) via direct regulation by oxidative-stress-associated molecules, and we hope that these results will bring new insights to better understand these fascinating enzymes. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished